This is a competitive renewal of R01 GM086703. Nicotinamide adenine dinucleotide (NAD), commonly known as an important redox cofactor in biology, are also used as a co-substrate by many NAD-consuming enzymes. These enzymes include CD38 (an NAD glycohydrolase and cyclase), poly(ADP-ribose) polymerases (PARPs), and sirtuins (NAD-dependent protein lysine deacylases). All these NAD-consuming enzymes have important biological functions and deregulation of them is associated with various human diseases, including cancer, diabetes, and neurodegeneration. However, many fundamental questions about the functions of NAD-consuming enzymes are poorly understood. The goal of this proposal is to develop and use chemical probes to help understand the function of these NAD consuming enzymes. In the last four years with NIH support, we have developed several NAD analogs to study these NAD- consuming enzymes and have made a number of important progresses. These include the development of covalent cell permeable probes for CD38, the development of a clickable NAD analog for labeling the substrate proteins of PARPs, and the discovery of Sirt5 as an NAD-dependent desuccinylase/demalonylase and Sirt6 as an NAD-dependent defatty-acylase. Building on these progresses, in this proposal, we will use the probes we already developed to understand the function of CD38 and PARPs, and to develop new probes for the study of PARPs and sirtuins. We believe our proposed studies will provide important insights to understand the biological functions of NAD-consuming enzymes, lead to the discovery of novel protein posttranslational modifications, and ultimately lead to the development of better therapeutics for treating human diseases involving NAD-consuming enzymes.